The present invention is related to detection and determination of protein S in biological fluids and to reagents for use therein. More specifically, free protein S is determined as a receptor/ligand complex formed between free protein S and a molecule comprising a ligand that binds specifically to protein S.
Protein S is a member of the naturally occurring anticoagulant protein C system (a part of the blood coagulation system) and acts as a cofactor to the activated state of protein C, APC (Activated Protein C), the other cofactor being intact Factor V. This system expresses anticoagulant action since APC acts so as to degrade the coagulation promoting Factors Va and VIIIa.
In human plasma, protein S circulates both as free protein and in complex with another plasma protein, the C4b-binding protein (C4BP) (Dahlbxc3xa4ck, Thromb. Haemostas. 1991, 66:49-61). Approximately 60% of the total protein S in plasma is bound to C4BP and it is noteworthy that this form of protein S is not functionally active as APC-cofactor. Thus, the binding of C4BP to protein S leads to a loss of the APC-cofactor function of protein S. The importance of protein S as an anticoagulant protein is illustrated by the association between protein S deficiency and thromboembolic disorders. Homozygous deficiency, which is extremely rare, gives a neonatal fatal disease, whereas heterozygous deficiency is a risk factor for venous thrombosis in adult life. Indeed, protein C deficiency or protein S deficiency is found in approximately 5 to 10% of all individuals exhibiting venous thrombosis.
An individual having protein S deficiency, thus, runs an increased risk of experiencing venous thromboembolic events. Accordingly, methods for determining blood or plasma levels of protein S have a potential clinical use. Particularly, methods for measurement of the levels of free protein S would be appreciated, since several investigators have shown that for the diagnosis of protein S deficiency, the level of free protein S should be measured rather than the level of total protein S or the bound form of protein S (Zxc3x6ller et al, Blood, 1995, 85:3524-3531). The reason for this is that higher sensitivity and specificity as regards the genetic defect causing protein S deficiency are achieved with the free protein S assays than with assays for measuring total protein S or bound protein S.
Previously known methods for determining free protein S are based on two different test principles, viz. differential polyethylene glycol precipitation properties and use of monoclonal antibodies, resp.
Methods using polyethylene glycol (PEG) to selectively remove bound protein S from a fluid comprising bound protein S and free protein S prior to measurement of protein S are based on the discovery that the complex bound form of protein S (PS:C4BP) precipitates already at a PEG concentration of approximately 3.75-5%, whereas most of the free protein S remains in solution. This principle has been used extensively in different commercially available protein S assays. Thus, in assays for free protein S, plasma samples are usually subjected to precipitation with PEG (3.75-5%) whereafter the protein S remaining in the supernatant after centrifugation is measured with immunological methods, such as ELISA, RIA or Laurell rockets. Such methods are disclosed in Am. J. Clin. Path. 94:176-186 (1990), Anal. Biochem., 10:358-361 (1985) and Blood, 67:504-508 (1986). However, these methods suffer from some disadvantages, mainly due to the PEG precipitation procedure. Thus, even when the PEG precipitation is highly standardized, this procedure is plagued by poor reproducibility and by its laborious and time consuming nature.
The second test principle mentioned above is based on use of monoclonal antibodies. Such monoclonal antibodies are specific for the free form of protein S, i.e. the epitopes for these antibodies are located at or close to the binding site for C4BP on the protein S molecule (Amiral et al, Blood Coag. Fibrinol. 1994, 5:179-186 and Wolf et al., Blood Coag. Fibrinol. 1994, 5:187-192).
C4BP, which as stated above binds to protein S and thereby reduces the amount of free protein S circulating in blood, is composed of approximately seven identical xcex1-chains, each of which contains a binding site for the complement protein C4b, and one single xcex2-chain. The xcex1-chains are linked in their C-terminal regions to each other and in addition to the single xcex2-chain. These seven xcex1-chains and the single xcex2-chain of the C4BP molecule are arranged like wheel-spokes to form a spider-like molecular structure (Dahlbxc3xa4ck and Stenflo in The molecular basis of blood disease, eds Stammatoyannopolous et al. WB Saunders 1994, p 599-627). The protein S binding site is known to be located on the single xcex2-chain and very recently (Hxc3xa4rdig and Dahlbxc3xa4ck, J. Biol. Chem. 1996, Volume 172, p. 20861-20867) the entire protein S binding site has been localized to the extreme N-terminal SCR-module (SCR stands for Short Consensus Repeat, which is a protein module containing approximately 60 amino acid residues) of the xcex2-chain. Although, this module has previously been proposed to contain the protein S binding site (Fernandez and Griffin, J. Biol. Chem. 1994, 269:2535-2540), it was not known before, that the entire protein S binding site is located in this first (extreme) SCR-module of the xcex2-chain.
The knowledge of the complex formation between protein S and C4BP, has been used to develop antibodies, which are specific for the free form of protein S. Thus, attempts have been made to raise antibodies that bind specifically to the region of protein S that is involved in the binding of C4BP, which antibodies obviously would only bind to free protein S, since in the C4BP-bound form of protein S, such binding sites in protein S, which are specific for these antibodies, are already occupied by C4BP. A prerequisite for development of antibodies with the said specificity is, however, specific knowledge of the C4BP binding site on the protein S molecule. Whereas this binding site has not been elucidated in detail in prior art, two areas in a large C-terminal module of protein S designated SHBG have been claimed to be involved. The first report suggested residues number 605-614 of mature protein S to be involved (Walker, J. Biol. Chem. 1989, 264:17645-17648) whereas another region comprising residues 413-433 (Fernandez et al., J. Biol. Chem. 1993, 268:16788-16794) has more recently been suggested to be important for the binding of C4BP to protein S.
In WO 93/01209 monoclonal antibodies directed to specific regions of mature protein S, contemplated to be involved in C4BP binding, are disclosed, which antibodies are useful in diagnostic methods and systems for purifying or detecting free protein S. Protein S polypeptides comprising these specific regions are also disclosed. These regions differ, however, from the C4BP binding regions disclosed below.
Moreover, assays for free protein S based on immobilized monoclonal antibodies directed to free protein S, which are used as immobilized antibody in standard ELISA (Enzyme Linked Immuno Sorbent Assay) to capture free protein S in plasma, have been described in the literature and are also commercially available from Stago (Amiral et al., Blood Coag: Fibrinol. 1994, 5:179-186, and Wolf et al., Blood Coag. Fibrinol. 1994, 5:187-192). In such tests, plasma dilutions in buffer containing calcium are incubated in microtitre plates containing monoclonal antibodies specific for free protein S, and, subsequent to washing steps, protein S bound to the monoclonal antibodies can be detected with the use of a second mono- or polyclonal antibody directed to protein S. However, such assays are extremely expensive. Furthermore, the antibodies used in these tests are not well characterized and they have not been raised specifically against any region of protein S suggested to be involved in the binding of C4BP to protein S. Rather, these antibodies have been raised against the entire protein S molecule, whereafter antibodies having specificity for free protein S have been selected.
It is an object of the present invention to provide a simple and reliable assay for determination of free protein S in biological fluids. According to the present invention, this object is achieved with an assay wherein a ligand that binds specifically to free protein S is added to a biological fluid comprising protein S to form a protein S/ligand complex where-after the level of free protein S is measured as the protein S/ligand complex formed in said fluid, and wherein said ligand is comprised of at least part of the C4b-binding protein (C4BP), C4BP being a naturally occurring ligand for free protein S, or a compound comprising an amino acid sequence homologous or analogous to the protein S binding site of C4BP or an amino acid sequence having essentially the same protein S binding properties as C4BP.
More specifically, the present invention is concerned with a method as defined in claim 1.
In accordance with a suitable embodiment of the present invention, the said ligand binding to protein S is derived from C4BP per se and comprises either the entire protein or a (poly)peptide fragment thereof having appropriate protein S binding capacity. Suitably, this fragment comprises the entire, or substantially the entire, protein S binding site of C4BP.
Although, it is well known that C4BP is a natural ligand that binds to protein S, prior to the present invention no one has suggested use of C4BP per se, or a fragment thereof, as a tool to measure in a biological fluid sample, the level of free protein S in presence of protein S bound as PS:C4BP in a test primarily based only on formation of a complex between free protein S and C4BP or appropriate fragments thereof.
According to the present invention it has, however, quite unexpectedly been found that C4BP can be used as a reagent component in an assay as disclosed herein for determining free protein S, since, unlike most proteins, C4BP is quite stable over time and is comparatively insensitive to heat. Moreover, the dissociation rate of the complex formed is sufficiently slow to enable measurement thereof, both qualitative and quantitative.
In connection with the present invention, the term xe2x80x9cligandxe2x80x9d is used to designate a molecular structure comprising an amino acid sequence that binds to an amino acid sequence of a receptor molecule, e.g. a protein peptide, polypeptide or the like, to form a molecular complex. In the present case, the receptor is comprised of free protein S. Thus, a ligand of the present invention can be comprised of an antibody paratope or a molecule comprising an amino acid sequence defining an antibody paratope, said molecule e.g. being an antibody or a fragment thereof.
According to one embodiment of the invention, the entire C4BP molecule, suitably in purified form, is used per se as a ligand to form a complex with free protein S, whereafter the complex formed, i.e. PS:C4BP, is measured in accordance with well known technique. It is of course necessary that this PS:C4BP complex formed in the assay can be distinguished from the PS:C4BP complex occurring naturally in blood. This can be achieved, e.g. by labelling and/or fixation of the C4BP used as ligand in the assay as is well known and will be disclosed more in detail further below.
Other embodiments of this invention are based on the knowledge of the exact localization of the protein S binding site in the C4BP molecule. Contrary to earlier reports, recently, this binding site has been found to occur in the extreme N-terminal SCR-module of the xcex2-chain of the C4BP molecule.
Accordingly, the present invention is also concerned with fragments, i.e. short polypeptides, of the C4BP molecule which comprise the protein S binding site of the C4BP molecule and can be used as ligands binding to protein S for the same purpose as the entire C4BP molecule with use of the same assay formats. As is well known, such fragments can be derived from C4BP, e.g. by enzymatic digestion thereof. After determination of the corresponding amino acid sequence such fragments or polypeptides may conveniently be produced with the use of conventional synthetic methods, such as a solid-phase Merrifield-type synthesis. Methods based on recombinant technology could also be used.
Other embodiments of the invention are related to the knowledge of the localization of the C4BP binding site in native protein S. Based on this knowledge protein S polypeptides comprising the said binding site can be obtained and used to raise antibodies, monoclonal or polyclonal, which are specific for this binding site and, thus, for free protein S. Obviously, such antibodies can be used to determine free protein S in the presence of protein S bound to C4BP since such antibodies will not bind to protein S complexed with C4BP, the corresponding antigenic determinant or epitope in complexed protein S being already occupied by the C4BP molecule.
Thus, further embodiments of the present invention are concerned with the above protein S polypeptides (PS polypeptides) comprising the C4BP binding regions of native protein S and with anti-PS polypeptide antibodies specific for these regions and, thus, inhibiting the binding interaction between C4BP and PS. Such polyclonal or monoclonal antibodies would inununoreact with free protein S and have, thus, potential use both as diagnostic reagents and as a therapeutic agent.
Accordingly, the present invention is also related to therapeutic compositions comprising said antibodies, which bind to free protein S and, thus, prevent inactivation of protein S through PS:C4BP complex formation. The present invention is also related to a therapeutic composition comprising a polypeptide or a monoclonal antibody of the present invention in an amount sufficient to inhibit the binding of free protein S to C4BP, which polypeptide or monoclonal antibody binds to and blocks the binding sites for free protein S comprised in C4BP, and a pharmaceutically acceptable carrier, excipient or diluent.
Still further embodiments of the present invention are concerned with diagnostic systems, suitably in kit form, for assaying in accordance with the present method, free protein S in a biological fluid, said systems comprising as separately packaged reagents, a ligand of the present invention and at least one further reagent, such as indicating means, buffer, etc, required to perform the assay. Suitably, these systems comprise all reagents necessary to perform the assay. Usually, instructions for use of the packaged reagents are included in these systems.
The present diagnostic systems and methods for measuring free protein S can be designed in a variety of conventionally used formats, preferably as direct immunoassays, such assays being based on the specific binding interactions between the C4BP binding region of protein S on one hand, and C4BP or fragments thereof or polypeptides comprising an amino acid sequence homologous or analogous to the protein S binding site of C4BP, or the present anti-PS-polypeptide antibody paratopes, on the other hand.
According to a further embodiment, such systems and methods can also be used to purify free protein S from fluid samples. Thus, the present invention is also related to a composition for purifying free protein S from an aqueous solution comprising a ligand of the present invention operatively linked to a solid carrier, and to a method for purifying free protein S from an aqueous solution comprising contacting the said solution with the said composition to form a protein S/ligand complex bound to the solid carrier, separating said complex from said solution and releasing protein S from the said complex.
In the following, the invention will be disclosed more in detail with reference to suitable embodiments thereof. Even though, the present invention is primarily concerned with protein S of human origin, the invention could also be applicable to protein S of other, e.g. bovine, origin.
As stated above, the present invention is primarily related to the binding interactions between protein S and C4BP. More specifically, the present invention is related to the use of ligands specific for free protein S, which ligands can be used to capture free protein S, e.g. in assays for free protein S, the expression xe2x80x9cfree protein Sxe2x80x9d being used as a distinction from protein S circulating in the living body in the form of a complex with C4BP. Whereas one embodiment of the invention is merely based on use of the naturally occurring protein C4BP per se as ligand, other embodiments of the present invention are related to detailed knowledge of such interactions, viz. specific knowledge of the localization and/or the specific amino acid sequence of each of the interactive binding sites in protein S and C4BP, which are involved in complex formation between Protein S and C4BP. Thus, the invention is concerned with use of C4BP related ligands as well as use of antibodies or fragments thereof binding to free protein S.
1) Use of Ligands Comprised of or Derived From C4BP
With respect to ligands comprising at least part of or the entire site of C4BP that binds to native protein S, although use of the entire C4BP molecule is convenient and constitutes a suitable embodiment of the present invention, advantages can also be expected to be achieved by use of fragments of C4BP, which fragments comprise the specific amino acid sequence, or at least part thereof, that binds to protein S. It has been shown recently (loc. cit.) that in C4BP, the native protein S binding site appears in the extreme (first) SCR-module of the xcex2-chain of the C4BP molecule. Thus, fragments which could be used as ligands in accordance with the present invention could be comprised of the intact xcex2-chain of the C4BP molecule or fragments of this chain, comprising or consisting essentially of the said N-terminal SCR module. Use of such polypeptide fragments instead of the entire protein could be advantageous with respect to ease of preparation of the ligand and improved affinity could be achieved if such fragments are used as ligands in the present method for measuring free protein S.
As mentioned above, such fragments could be prepared by means of conventional peptide synthesis or methods based on recombinant technology, whereas use of C4BP per se usually encompasses isolation of C4BP from plasma in the form of the PS:C4BP complex, separation of C4BP from protein S and further purification. Appropriate fragments of C4BP, could also be derived from blood, for instance by enzymatic cleavage of C4BP obtained from blood, as disclosed above.
Moreover, use of recombinant technology opens up possibilities to design C4BP-like ligands having properties, which make such ligands specifically useful as catching ligands for free protein S. Thus, with use of recombinant technology, a hybrid molecule between the two types of chains, i.e. the xcex1-chain and the xcex2-chain, of the C4BP has been produced. In this construct, the extreme N-terminal SCR of the xcex2-chain is replacing the corresponding module of the xcex1-chain. The recombinant product obtained is a C4BP-like molecule having multiple disulphide-linked subunits, each of which contains a protein S binding site.
Even though it is possible to design C4BP-like molecules which could be very efficient as catching ligands in assays for free protein S, suitable embodiments of the invention are based on use of the entire C4BP molecule or, rather, C4BP species comprising the xcex2-chain, i.e. C4BPxcex2, as ligands in such assays. Since the binding site on C4BPxcex2 binds protein S with very high affinity (KD=0.1 nM) in the presence of physiological calcium concentrations, the association rate constant in the presence of calcium ions being high (almost 105 Mxe2x88x921 s-1) and the dissociation rate constant being low (approximately 5xc3x9710xe2x88x924 s-1), C4BPxcex2 (containing an unoccupied protein S binding site on the xcex2-chain) is a highly specific and efficient ligand for free protein S and is able to specifically bind to free protein S in a solution containing both free protein S and PS:C4BP complexes.
It is, of course, essential that C4BP used as ligand in the present assay is substantially comprised of C4BP species containing the xcex2-chain and, thus, the protein S binding site. This means that C4BP used in the present method is substantially comprised of its major isoform C4BPxcex2 having seven xcex1-chains and one xcex2-chain as stated above, and that its minor isoform lacking the xcex2-chain is absent or present in a low proportion.
In accordance with a suitable embodiment of an assay for free protein S of the present invention, C4BPxcex2 is immobilized on a carrier, e.g. a microtiter plate, and is contacted, i.e. incubated, with a solution containing both free protein S and PS:C4BP complex to specifically bind to and, thus, extract free protein S from said solution, whereafter protein S, which is bound to the immobilized ligand, can be detected with mono- or polyclonal antibodies specific for protein S.
The high association rate constant in the presence of calcium allows very short incubation times to be used for this primary capturing. Suitably, a few washing steps are performed prior to the incubation with the said mono- or polyclonal antibodies. In principle, any antibody specific for protein S may be used. However, according to a suitable embodiment of the present method a monoclonal antibody is used, which has some unique properties, which makes it most suitable. This antibody, which is designated HPS54, has been characterized (Dahlbxc3xa4ck et al., J. Biol. Chem. 1990, 265: 8127-8135) and possesses unusually high affinity for protein S. Its epitope is located in the first EGF-like domain of protein S, i.e. it is distinct from the binding site for C4BPxcex2, which is located in the SHBG region, and calcium is required to achieve the high affinity of the binding of the said antibody to protein S. These unique properties make this antibody a suitable reagent to detect the protein S, which has been retained by the immobilized C4BPxcex2. To enable detection of protein S bound to the immobilized ligand, which protein S/ligand complex also carries bound HPS54 monoclonal antibody, HPS54 is either directly labelled to enable detection thereof or detected with secondary steps, such as secondary antibodies against this monoclonal antibody.
The method disclosed above using C4BP as a ligand is only given to illustrate the invention. The invention is not restricted to this embodiment but there is an almost unlimited number of possible ways to use the described principle for measurement of free protein S and a number of different designs of the assay principle are possible. Thus, modifications and further embodiments of the invention are obvious for he skilled artisan.
2) Use of Ligands Comprised of Antibodies Specific For Free Protein S
According to the present invention, the ligands used to capture free protein S can also be comprised of antibodies specific for free protein S. Thus, the present invention is related to such antibodies which have been raised directly against a region of protein S, which has been found to be involved in the binding interactions between protein S and C4BP.
Such antibodies are obtained with the use of the present PS polypeptides comprising the said specific regions, said polypeptides being used to prepare an inoculum, which is used to obtain the antibodies having the desired specifity, e.g. by administration (immunization) of an appropriate animal.
These PS polypeptides comprise regions of mature protein S that differ from such regions of protein S, that in prior art have been suggested to be involved in the binding of C4BP.
In accordance with the present invention, the regions of protein S, which have been found to be involved in the binding of C4BP, and which, thus, when present in an antigen, e.g. a polypeptide, are potentially useful as immunogens or antigens to raise antibodies specific for free protein S, all include the amino acid residues 447-460 of mature protein S, represented by the formula SGIAQFHIDY NNVS.
In general terms, the present invention is, thus, concerned with PS polypeptides, said polypeptides comprising at least amino acid residues 447-460 of mature protein S. Optionally, said polypeptides may comprise additional N-terminal and/or C-terminal amino acid residues that differ from the corresponding flanking residues of protein S. The PS polypeptides could also comprise an N-terminal or C-terminal part of the 447-460 amino acid sequence and resp., additional N-terminal or C-terminal flanking residues corresponding to those of mature protein S, or could comprise the entire 447-460 amino acid sequence with additional flanking residues at both ends, which flanking residues correspond to those of mature protein S. Illustrative of such PS polypeptides are polypeptides comprising amino acid residues 439-460, 447-468, and 435-468, resp., (Table 1). The inventors have shown that synthetic polypeptides corresponding to the above sequences are capable of inhibiting protein S-C4BP interaction, whereas synthetic polypeptides corresponding to residues 405-437 and 595-628 (Table 1), which in prior art have been suggested to be involved in the binding of C4BP, did not show any inhibitory effect, not even if used in excess (2000xc3x97) over protein S. Thus, the present polypeptides comprise an amino acid residue sequence corresponding to the amino acid residues 447-460 of mature protein S and optionally additional flanking residues of mature proteins at one or both ends but suitably do not extend beyond amino acid residue 438 of mature protein S in their N terminus or amino acid residue 526 of mature protein S in their C terminus. Apart from being derived from protein S, the present polypeptides can also be prepared with conventional polypeptide synthesis.
The above amino acid numbering of protein S corresponds to the conventional numbering used e.g. in Lundwall, xc3x85. et al., Proc. Nat""l Acad. Sci. USA, vol. 83, p. 6716-6720 (human protein S) and Dahlbxc3xa4ck, B. et al., Proc. Nat""l Acad. Sci. USA, vol. 83, p. 4199-4203 (bovine protein S), which references disclose the cDNA and the amino acid sequence of protein S.
Accordingly, a suitable embodiment of the present invention is directed to antibodies, polyclonal or, preferably, monoclonal, that have been raised against the above PS polypeptides and, thus, are capable of immunoreacting with the above amino acid residue sequences of protein S, which have been found to be involved in high affinity binding of C4BP to free protein S. As explained above, such antibodies will be specific for free protein S and will not bind to protein S complexed with C4BP.
The present antibodies are referred to as anti-PS antibodies and are characterized by immunospecifity for free protein S. In addition, they can be expected to be capable of inhibiting binding of protein S to C4BP and, thus, to be useful as a drug to enhance the amount of free protein S in blood.
Suitable anti-PS antibodies of the present invention are capable of immunoreacting with a polypeptide comprising the amino acid residues 447-460 of mature protein S, said antibodies also immunoreacting with the same amino acid sequence in free protein S.
The present antibodies can be produced in accordance with commonly known methods with use of commercially available protocols. Generally, an animal, preferably a mammal, is inoculated, e.g. injected, with a PS polypeptide of the present invention comprising amino acid residues 447-460 of mature protien S and used in an amount sufficient to induce production of antibodies in said animal. Subsequently, the antibodies thereby produced are collected from the animal, suitably in serum, ascites or some other body fluid, or an antibody-producing organ, such as spleen, is used to produce antibodies with recombinant technique.
Those antibodies, that have the desired immunospecifity are preferably isolated from e.g. the body fluid, suitably by immunoaffinty chromatography, or with other well known techniques. If immunoaffinity chromatography comprising solid phase-affixed inmmunizing polypeptide is used to purify the antibodies, their specifity could be enhanced. Such immunoaffinity chromatography comprises contacting the antibodies with solid phase-affixed immunizing polypeptide, a solid phase-affixed immunocomplex being formed and, subsequent separation of the antibodies from this immunocomplex.
Since the PS polypeptides used to immunize the animal are short polypeptides, they are preferably included in the inoculum linked to a carrier to form a conjugate. Use of such conjugates are preferred for peptides comprising about 35 amino acids or less. Suitable carriers are well known in the art and comprise keyhole limpet hemocyanin (KLH), hemocyanin from horseshoe crab (Limulus polyphemus), edestin, thyroglobulin, albumins and the like.
To assist in linking the polypeptide to the carrier, the polypeptide may comprise additional amino acid residues added to the amino- or carboxy-terminus of the polypeptide. Suitably, cystein residues are added and a carrier is used comprising free cystein residues so that a polypeptide/carrier conjugate is obtained by the formation of disulphide bonds. Such added cystein residues may also assist in performing the above immunoaffinity purification, e.g. by binding the immunizing polypeptide comprising added cystein residues to an affinity matrix comprising free cystein residues, e.g. ThiolSepharose(copyright) from Pharmacia Fine Chemicals.
The present antibodies can be polyclonal or monoclonal, monoclonal antibodies usually being preferred. The expression xe2x80x9cantibodyxe2x80x9d is intended to refer not only to the entire antibody but also to appropriate fragments thereof. Monoclonal antibodies contain only one single species of antibody combining site, or paratope, capable of immunoreacting with a particular epitope. However, a monoclonal antibody may comprise more than one specific antibody combining site, such antibodies being polyspecific, e.g. bispecific. Suitably the present monoclonal antibodies are monospecific and comprise a single paratope specific for the present PS polypeptides and are, thus, also specific for free protein S.
The preparation of monoclonal antibodies is well known in the art and was first disclosed by Koehler and Milstein, Nature, 256: 495-497, 1975. As disclosed in this reference, monoclonal antibodies are produced by clones of one single cell designated hybridoma. These hybridomas are formed by fusion of an antibody-producing cell, usually lymphocytes, with a myeloma or other self-perpetuating cell line and they secrete antibodies into the supernatant of the hybridoma cell culture. To produce the present hybridomas, lymphocytes are used, which have been collected from an animal previously hyperimmunized with the present PS polypeptide as antigen. The present invention is also related to hybridoma cells and cell cultures containing such hybridoma cells that produce a monoclonal antibody of present invention.
Preferably, the polyclonal or monoclonal antibodies of the present invention immunoreact with the present PS polypeptides disclosed in the experimental part of this disclosure, and, thus, immunoreacts with free protein S with high specifity.
In accordance with a suitable embodiment of the present invention such antibodies can be used in a test system, e.g. a diagnostic kit, which is intended to be used in an ELISA format to detect the quantity of free protein S in a fluid sample, such as blood, serum, or plasma, wherein this antibody is bound to a solid phase and an enzyme-antigen conjugate is used to detect and quantify the amount of antigen, i.e. free protein S, in a sample.
As stated above, the present antibodies and the C4BP related ligands can be used in a diagnostic system for assaying free protein S. In accordance with the present invention, such systems usually also include indicating means to facilitate determination, qualitative or quantitative, of the receptor/ligand complex formed in the assay of the present invention. Such indicating means may be used in addition to fixation of the ligand to a substrate, or as an alternative enabling assays of said complex in free form, i.e. not bound to a solid carrier, matrix, or the like.
Such indicating means or labels are comprised of single atoms or molecules, that can be linked to or incorporated in the present ligand, or used separately, and that are involved, either directly or indirectly in the production of a detectable signal to indicate the formation of the present receptor/ligand complex. Additional reagents may be required, e.g. in connection with enzyme labels, the corresponding substrate being required to visualize the signal. Useful indicating means or labels are well known within this field of art and e.g. include chromogenic, fluorogenic and cheriluminogenic labels, suitably fluorogenic labels, such as fluorescein isocyanate (FIC). Other labels, which can be used, are enzymes, such as horseradish peroxidase (HRP), and radioactive isotopes, such as 125I.
Suitably, the present ligand is affixed, e.g. by absorption, to a solid matrix. Useful solid matrices are well known in the art and are composed of water insoluble materials, such as cross-linked dextran available under the trademark Sephadex from Pharmacia Fine Chemicals; agarose; beads of polystyrene having a diameter of about 1 xcexcm to about 5 mm and available from Abbott Laboratories of North Chicago, Ill.; polyvinyl chloride, polystyrene, cross-linked polyacrylamide, nitrocellulose- or nylon-based webs, such as sheets, strips or paddels; or tubes, plates or the wells of a microtiter plate, such as those made from polystyrene or polyvinyl chloride.
The reagent components of the diagnostic system described herein can be provided in solution or as a liquid dispersion. However, suitably, they are provided as a substantially dry powder, preferably in lyophilized form. If an enzyme is used as indicating means, the corresponding substrate can also be provided in a separate package of the system. A solid support, such as a microtiter plate as mentioned above and one or more buffers can also be included as separately packaged elements in the present diagnostic system.
Suitable embodiments of the present invention are explained in more detail in the illustrative examples and with reference to the accompanying drawings.